SIGNAL TRANSDUCTION IN PANCREATIC β-CELLS: REGULATION … · signal transduction is paralleled by...

[Frontiers in Bioscience 2, d160-172; March 15, 1997]

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SIGNAL TRANSDUCTION IN PANCREATIC ββ-CELLS: REGULATION OF INSULINSECRETION BY INFORMATION FLOW IN THE PHOSPHOLIPASE C/PROTEINKINASE C PATHWAY

Walter S. Zawalich1, Marc Bonnet-Eymard, and Kathleen C. Zawalich

Yale University School of Nursing, 100 Church Street South, New Haven, CT 06536-0740 USA

TABLE OF CONTENTS

1. Abstract2. Introduction3. Multiple effects of glucose on the pancreatic β-cell

3.1 Acute regulation of insulin release3.2 Fail-safe regulation of insulin secretion and time-dependent potentiation3.3 Time-dependent suppression of insulin release

4. Glucose-induced insulin secretion: role of phospholipase C activation4.1 Effects of glucose on PLC-mediated phosphoinositide (PI) hydrolysis4.2 PLC isozymes in islets and their activation

5. Glucose-induced insulin secretion: role of protein kinase C activation5.1 Protein kinase C activation in freshly studied rat islets5.2 Protein kinase C activation and glucose-induced insulin secretion from cultured islets5.3 Insulin secretion from PKC-depleted islets

6. Species differences in glucose-induced insulin secretion6.1 Glucose-induced insulin release from mouse islets6.2 PLC activation in mouse islets6.3 Time-dependent potentiation in mouse islets6.4 Glucose fails to induce time-dependent suppression in mouse islets

7. The beta cell, obesity and NIDDM8. Summary and Perspectives9. Acknowledgements10. References

1. ABSTRACT

The physiologic regulation of glucose-induced insulin secretion is dependent upon theactivation of information flow in the phospholipase C(PLC)/protein kinase C (PKC) signal transductionsystem. In both rat and human pancreatic β-cells,glucose has several time-dependent effects onsecretory responsiveness including the regulation ofbiphasic insulin secretion, time-dependentpotentiation and time-dependent suppression.PLC/PKC activation has been implicated in all threeresponse patterns. Islets of Langerhans contain thethree major PLC isozyme classes (β1, γ1 and δ1) andthe available evidence suggests that one class isactivated by fuel secretagogues and another byneurohumoral agonists. The expression and activationof PLC is labile. When rat islet are cultured for short___________________________________________Received 2/24/97; Accepted 2/27/971 To whom correspondence should be addressed at,Yale University School of Nursing, 100 Church StreetSouth, New Haven, CT 06536-0740 USA. Tel #: 203-785-5522; Fax #: 203 785-6455. Email: [email protected]; marc.bonnet-eymard@yale. edu;[email protected]

periods, the content and activation of PLC in responseto glucose decreases and this biochemical defect insignal transduction is paralleled by significantreductions in glucose-induced insulin secretion.Similar defects are observed when human islets arecultured as well. Mouse islet responses to glucosestimulation differ in several major and dramatic waysfrom rat and human islet responses. When stimulatedby 15mM glucose, mouse islets fail to develop arising second phase secretory response, they fail toexhibit either time-dependent potentiation or time-dependent suppression to the hexose. Biphasicinsulin secretion can be evoked and time-dependentpotentiation can be induced in mouse islets bycarbachol, an agonist that activates an isozyme ofPLC distinct from that activated by glucose. Thesedivergent response patterns are attributable to theunderexpression in mouse islets, when compared torat islets, of a fuel-sensitive PLC isozyme. Whentaken in their entirety, the experimental evidencesuggests that the activation of PLC is an essentialcomponent in the physiologic regulation of insulinsecretion and that disordered activation of the enzymeculminates in disordered insulin release.

Signal transduction in pancreatic ββ-cells

161

Figure 1. Biphasic Glucose-induced Insulin Secretionfrom Perifused Rat Islets but not Perifused MouseIslets. Groups of rat (closed circles) or mouse (opencircles) islets were collagenase isolated and perifusedwith 3mM glucose (G3) for 30 minutes prior tostimulation with 15mM glucose (G15). Note thecharacteristic biphasic insulin secretory responsecharacterized by a rising second phase evoked fromrat islets but the flat sustained response of mouseislets.

2. INTRODUCTION

The maintenance of glucose homeostasisdepends upon a well orchestrated series of hormonal,metabolic, ionic and second messenger signalingevents. At the level of the pancreatic β-cell, theseprocesses ensure that the release of insulin will becommensurate to satisfy peripheral tissuerequirements for the hormone and, as a consequenceof its pleiotropic effects on a variety of target tissues,maintain fuel homeostasis. Failure at any level in thiscascade of β-cell signals puts the organism at risk forglucose intolerance, a pathognomonic clinicalcomplication we commonly associate with Type IInoninsulin dependent diabetes (NIDDM). It is not thepurpose of the present review to revisit the multiplesignaling events which occur in peripheral tissuessuch as liver and muscle which respond to insulin orto the postulated gut (incretin) factors, such asglucagon-like peptide-1 or gastric inhibitory peptide,which participate in the physiologic regulation ofinsulin secretion. Rather the scope of this review isintentionally narrow with a primary focus on howglucose and other secretagogues activate thepancreatic β-cell and the role of increasedinformation flow in the phospholipase C (PLC)/protein kinase C (PKC) signaling system in theregulation of insulin secretion. This is a mostdynamic area of investigation and it is one that is notwithout significant controversy as well. Reasons forthe discrepancies which exist in the literature will beaddressed so that the reader may form their ownconclusions as to the relevance of the reported resultsto the physiologic regulation of insulin secretion. Atthe outset, however, it is important to review the

multiple and diverse effects that glucose has on thepancreatic β-cell. The comments that follow will dealprimarily with the time-dependent actions thatglucose exerts on insulin secretion. It should be bornein mind, however, that additional effects on insulinbiosynthesis and β-cell enzyme induction have alsobeen described.

3. MULTIPLE EFFECTS OF GLUCOSE ONTHE PANCREATIC ββ-CELL

3.1 Acute Regulation of insulin release.The release of insulin from the pancreatic

β-cell is dependent upon the interaction of a varietyof signaling molecules with β-cell signal transductionsystems

(1-3)

. It is commonly accepted that glucose is the primaryphysiologic regulator of β-cell sensitivity and that itexerts multiple effects on β-cell response patterns.At postprandial concentrations, it independently


162

increases insulin secretion. Sustained, short-term (1hour or less) glucose stimulation of rat islets resultsin a biphasic insulin secretory response (Figure 1)characterized by a transient first phase of release anda slowly rising and sustained second phase responsethat is, depending on the preparation and the glucoselevel, 30-60 fold greater than prestimulatory releaserates

(4-8)

. A qualitatively

(9,10)

and quantitatively


163

(11,12)

similar biphasic insulin secretory response tosustained glucose stimulation has been noted fromhuman β-cells studied with the hyperglycemic clampmethodologies, observations which lend credence tothe concept that the biochemical signaling eventswhich regulate insulin secretion from rat β-cells aresimilar, if not identical, to the events which occur inhuman β-cells. Thus it is not unreasonable toextrapolate findings made with rat islets to humanislets. As will become evident later, similarextrapolations with mouse β-cells must be cautiouslymade since pronounced species differences, whichhave been ignored in many instances, exist in thepatterns of glucose-induced insulin secretion frommouse islets.

3.2 Fail-safe regulation of insulin secretion andtime-dependent potentiation

In addition to stimulating a biphasic insulinsecretory response, glucose also performs the role of a

fail-safe regulator of secretion. For example, atglucose levels greater than 6mM acetylcholine

(13)

or cholecystokinin (CCK)


164

(14)

are potent stimulants for secretion but athypoglycemic levels they fail to activate insulinexocytosis from the β-cell. This, so-called permissiveeffect of glucose, is thought to be a result of glucosemetabolism and the provision of adequate amounts ofATP to support a secretory response

(1)

. The fact that other nutrient secretagogues that arewell metabolized supply the same permissive signalssupports this concept


165

(15)

. Glucose stimulation of the β-cell also sensitizes it tosubsequent restimulation with a variety of agonists

(4, 7, 16,17)

. This response is induced by prior short-termstimulation with glucose and manifests itself in theheightened release of insulin during subsequentrestimulation. Termed time-dependent potentiation(TDP), this phenomenon can be induced by agonistsas structurally diverse as acetylcholine


166

(18)

, cholecystokinin

(19)

, α-ketoisocaproate

(20)


167

, glyceraldehyde

(21)

, monomethylsuccinate

(15)

, methyl pyruvate


168

(22)

and, perhaps most importantly, by the PKC activatortetradecanoyl phorbol acetate (TPA)

(23,24)

. TDP may play a particularly important role incephalic phase insulin secretion where vagally-derived acetylcholine may be responsible for itsinduction


169

(25,26)

.

3.3 Time-dependent suppression of insulin releaseIn addition to acutely regulating insulin

secretion, acting as a fail-safe modulator when isletsare stimulated with neurohumoral agonists or gutpeptides like CCK, and inducing TDP, glucose exertsanother additional important effect on the β-cell.When chronically stimulated with high glucose, β-cell insulin secretion fails. Termed the third phase ofsecretion by Grodsky and coworkers

(27,28)

, β-cell desensitization is an inevitable consequenceof sustained high glucose exposure


170

(6)

. This process, also referred to as time-dependentsuppression (TDS) of release

(29)

, may be analogous to the β-cell glucose toxicitynoted in human diabetes and other animal models ofglucose intolerance


171

(30-32)

. The capacity to induce TDS of insulin secretion isnot confined to glucose alone. It can be induced bysustained exposure to agonists as diverse ascholecystokinin

(33)

, glucosamine


172

(34)

, carbachol

(35)

and forskolin

(36)


173

. What suppressed islets have in common is theirinability to respond, in terms of increases in bothPLC-mediated PI hydrolysis and insulin secretion,when subsequently challenged with high glucose

(29)

. It should be emphasized that secretory failure is notthe result of insulin depletion since desensitized isletsstill contain large amounts of insulin

(6, 35,37)

and they will respond to an agonist which bypassesthe lesion in PLC activation


174

(15,34)

.

4. GLUCOSE-INDUCED INSULIN SECRETION:ROLE OF PLC ACTIVATION

4.1 Effects of glucose on PLC-mediatedphosphoinositide (PI) hydrolysis

The ability of glucose stimulation toincrease insulin secretion has been the subject ofintense experimental analysis. In large part, this isdue to the realization that the emergence of NIDDMis due to β-cell decompensation and their inability torespond to glucose stimulation

(30,38)

. Glucose must be metabolized to augment secretionand the consensus is that the increase in ATP levelsor the ATP/ADP ratio results in the closure of ATP-sensitive K+ channels, cell depolarization and theinflux into the β-cell of calcium via voltage-regulatedCa2+ channels


175

(1,39)

. While glucose-dependent insulin secretion clearlydepends on extracellular calcium influx into the β-cell

(40-42)

, this event alone is insufficient to explain the largeand rising biphasic insulin secretory response whichoccurs in response to the hexose. For example, β-celldepolarization with high levels of K+ and the ensuingsustained increase in intracellular calcium results in alarge first phase insulin secretory response with littlesustained stimulatory effect on release


176

(43-45)

. Other signals dependent on glucose metabolismmust be generated and, since the mitochondrial fuelsand insulin secretagogues α-ketoisocaproate andmonomethylsuccinate duplicate many of the actions ofglucose

(15,20)

, a mitochondrial signal in addition to calcium hasbeen postulated


177

(46)

. Moreover, mitochondrial signals and calcium arealso essential for the full activation of PLC by glucose

(45)

leading to the conclusion that the activation of PLCmay play an important role in the regulation ofglucose-induced insulin secretion.

A variety of techniques have been employedto assess the impact of glucose stimulation on PLC-mediated phosphoinositide hydrolysis. Mostinvestigators incubate islets, usually isolated fromrats, in myo-[2-3H]-inositol for various lengths oftime. This label is exclusively incorporated into thefamily of phosphoinositide lipids. After washing toremove unincorporated label, the islets are thenstimulated with various agonists and theaccumulation of labeled inositol phosphates (IPs),derived from phosphoinositide precursors, serves asthe index of PLC activation (Figure 2)


178

(47,48)

. Alternatively islets can be perifused after labelingand the efflux of free [2-3H]-inositol measured in theperifusate along with insulin

(14, 42,49)

. Similar results have been obtained with bothapproaches and the conclusion reached is that glucoseactivates PLC, a response dependent upon bothglucose metabolism


179

(16)

and calcium influx

(45)

. The precise identity of the metabolic signal hasremained elusive but it can be generated by bothglycolytic and mitochondrial fuels

(50,51)


180

.

4.2 PLC isozymes in islets and their activationAn additional level of complexity has been

added by nature to the activation of PLC. This has todo with the recent demonstration that rat islets

(52)

like many other tissues

(53)


181

express more than one type of PLC. β-cells containthe three major isozymes of PLC (β1, γ1 and δ1) andthe available evidence suggests that different classesof insulin secretagogues activate distinct isozymes ofPLC

(52, 54,55)

. For example, nutrient secretagogues activate theenzyme by increasing intracellular calcium andgenerating an as yet to be identified mitochondrialsignal

(41,45)

. Evidence for this concept derives from the fact thatglucose must be metabolized to increase PIhydrolysis, that its effects on PLC activation can beduplicated by both glycolytic and mitochondrialsubstrates and that the full activation of PLC requiresboth metabolism and calcium influx into the β-cell.While calcium contributes to the activation of a fuel-regulated PLC, sustained


182

Figure 2. Dose-response Effects of GlucoseStimulation on Phosphoinositide Hydrolysis. Groupsof rat (closed circles) or mouse (open circles) isletswere incubated for three hr in 3H-inositol, washed toremove unincorporated label and then stimulated withvarious levels of glucose. The inositol phosphates (IP)accumulating during a 20 minute (rat) or a 30 minute(mouse) stimulatory period with the hexose areshown here plotted against the glucose level. Alsoindicated is the IP response of mouse islets (opencircle) stimulated with the combination of 15mMglucose plus 100µM carbachol demonstrating thatwith the proper agonist a significant IP response canbe generated.

increases in intracellular calcium induced bydepolarizing levels of potassium result in only smallincrements in insulin secretion and IP accumulation

(45)

. Furthermore blocking calcium influx reduces butdoes not abolish glucose-induced IP accumulation

(41)


183

. The simplest interpretation of these and other datais that at least two signals--calcium andmetabolically-derived factor- -are involved in theactivation of the nutrient-regulated PLC.

The concept that neurohumoral agonistssuch as acetylcholine or its nonhydrolyzable analoguecarbachol activate an isozyme of PLC different fromthat activated by nutrients is supported by both thecalcium and metabolic independence of neurohumoralagonists on IP accumulation

(47, 56,57)

. Most importantly maximal stimulatory levels ofglucose and these neurohumoral agonists interact inat least an additive fashion to increase IPaccumulation

(54,57)


184

. However, perhaps the most cogent data supportingthe concept that different isozymes of PLC regulatethe responses to fuel and neurohumoral agonists onPLC-mediated PI hydrolysis and insulin secretioncomes from studies where the responses of rat isletswere compared to those from mouse islets, the twomost commonly employed species used to unravel thecomplex maze of signaling events which regulateinsulin secretion

(52)

. Most importantly the divergent effects of glucose onthe activation of PLC in these species was paralleledby a marked divergence in the insulin secretoryresponse (Figure 1) to the hexose as well (See below,Section 6).

5. GLUCOSE-INDUCED INSULIN SECRETION:ROLE OF PROTEIN KINASE C ACTIVATION

5.1 Protein kinase C activation in freshly studiedislets

The response of the perfused rat pancreaspreparation to glucose stimulation is a biphasicrelease of insulin. In freshly-isolated rat islets, theaddition of a stimulatory glucose level (15-20mM)also results in a dramatic and sustained biphasicinsulin secretory response

(58,59)


185

(See also Figure 1). In particular, the magnitude ofthe second phase response is 30-40 fold greater thanprestimulatory secretion rates. The calcium-dependent activation of PLC also occurs and IPlevels, used as a surrogate marker for PLC activation,increase 300-400%

(41,47)

(See Figure 2). Since, in addition to the inositolphosphates, PLC-mediated PI hydrolysis results in thegeneration of diacylglycerol

(60)


186

, the endogenous activator of PKC

(61)

, the activation of this enzyme might be anticipated aswell. In fact, studies in vivo or in vitro with theperfused rat pancreas

(62)

or perifused rat islets


187

(63)

have demonstrated that PKC translocates from apredominantly cytoplasmic compartment to amembrane one, a response often used to indicate itsactivation. Furthermore, the phosphorylation state ofthe MARCKS protein, an established protein targetfor activated PKC, is increased

(64)

. Blocking PKC activation with low levels of theinhibitor staurosporine reduces both MARCKSphosphorylation


188

(64)

and second phase secretion from rat islets

(65)

consistent with an important contributory role forPKC in determining the effects of glucose on the β-cell. PKC activation by glucose or TPA also increasescAMP levels in islets, providing an additional secondmessenger mechanism to amplify insulin secretion

(66,67)


189

. However, based on studies with cultured islets, theconclusion has been reached that PKC activation maynot be involved in glucose-induced insulin secretion

(68,69)

. The reasons for this continuing controversy and apossible resolution to this issue deserve furthercomment.

5.2 PKC Activation and Glucose-induced InsulinSecretion from Cultured Rat Islets

In a series of early studies using the phorbolester TPA, an exogenous PKC activator, to stimulatethe β-cell we suggested that the activation of PKCmay be playing an important role in the rising secondphase secretory response evoked by glucose

(70)


190

. We based this suggestion on the slowly rising andsustained release of insulin that followed β-cellexposure to the phorbol ester

(67,70)

, a response that qualitatively was somewhatreminiscent to that seen with glucose stimulation.This concept- that PKC activation regulated at leastin part glucose-


191

Figure 3. Glucose-induced Insulin Secretion isImpaired from Rat Islets after Short Term Culture.Two groups of rat islets were studied. One group(closed circles) was perifused with 3mM glucose(G3) for 30 min and with 15mM glucose (G15)immediately after isolation. The second group (opencircles) was cultured 22-24 hours in CMRL-1066medium supplemented with 10% fetal calf serumprior to perifusion. The glucose level of the mediumwas 5-5.5mM. Note the dramatic reduction insecretion from cultured islets.

induced insulin secretion-has been both challengedand supported. Most of the negative data concerningthe role of PKC activation has been generated withislets cultured for various periods of time afterisolation. However, the activation of PKC is not theonly aberration cultured islets demonstrate. Whenstimulated by high glucose, the accumulation of IPs inone-day cultured islets is reduced

(71)

, high glucose fails to translocate PKC to themembrane

(68)


192

, islet content of PLCδ1 falls by about 50%(unpublished observation), residual insulin release isimmune to inhibition by staurosporine

(72)

and, when primed by high glucose, time-dependentpotentiation is not induced (unpublished observation).Most telling, however, is the minimal insulinsecretory response observed when cultured rat isletsare stimulated with glucose

(68,71)


193

. The magnitude of this response, 4-5 fold abovebasal stimulatory rates (See Figure 3), stands incontrast to the robust 30-40 fold increase seen fromthe perfused pancreas preparation or from our freshly-isolated rat islets. The decline in islet responsivenessto glucose observed after culturing is not confined torat islets. Cultured human islets suffer the samefunctional decline. For example the human β-cellresponse to glucose stimulation in vivo is a 10-20 foldincrease in second phase insulin secretion rates

(10,12)

. In sharp contrast the second phase insulin secretoryresponse to 28mM glucose from cultured humanislets is flat

(72)


194

, less than 2-fold greater than prestimulatory secretionrates and is paralleled by the minimal activation ofPKC as well

(72)

.

5.3 Insulin secretion from PKC-depleted rat isletsIn an attempt to establish the involvement

of PKC activation in the sequence of biochemicalevents which regulate physiologic glucose-inducedinsulin secretion from rat β-cells, several groups havecultured islets in the presence of TPA to deplete theislet of PKC. There are actually at least three issueshere but only one is usually addressed. First is theeffect of culturing which alone impairs glucose-induced insulin secretion

(71, 73,74)


195

irrelevant of any other additions to the culturemedium. The second is the added effect of sustainedPKC stimulation on an islet whose secretory integrityis deteriorating as a result of culturing. Finally, thetacit assumption that only PKC content is altered andthat all of the protein substrates for PKC remain in abasal or unstimulated state is unwarranted. SincePKC activation exerts long term effects on β-cellsensitivity, effects presumably mediated by thesustained phosphorylation of PKC substrates

(75)

, the fact that PKC is depleted does not address thepossible contribution of these phosphoproteins toinsulin release

(76)


196

. To emphasize the problem with cultured islets oneonly has to study the secretory data from one report

(69)

often cited as evidence that PKC activation is notinvolved in the physiologic regulation of glucose-induced insulin secretion. In this study, the insulinsecretory response after 20-24 hour of culture fromcontrol islets stimulated with 5.6mM glucose was0.44 ng/h per islet. The insulin secretory response ofcultured control islets to 20mM glucose increased to0.75 ng/h per islet and less than a doubling of insulinoutput occurred. This observation stands in sharpcontrast to the 30-fold increase in release rates weand others have obtained with freshly isolated isletsand casts a considerable doubt on the physiologicsignificance of any further observations made withsuch profoundly impaired islets irrelevant of theirPKC content.

To conclude that PLC/PKC activation is notinvolved in the physiologic regulation of insulinsecretion from a preparation (cultured islets) whichdiffers in several substantial ways from the responsesof fresh islets is inappropriate. We have repeatedmany of these studies with cultured rat islets andarrived at the same conclusion


197

(71)

. In islets whose insulin secretory responsiveness toglucose is dramatically impaired by culturing, theactivation of PLC/PKC signaling events may not beinvolved in the minimal insulin secretory responsesobserved. We

(54,55)

and others


198

(39)

have dealt with this issue in several articles.However, studies with cultured islets have reinforcedour conviction that information flow in the PLC/PKCsignaling system is an essential component ofphysiologic glucose-induced insulin secretion.

6. SPECIES DIFFERENCES IN PLCACTIVATION, GLUCOSE-INDUCED INSULINSECRETION, TIME-DEPENDENTPOTENTIATION AND TIME DEPENDENTSUPPRESSION

6.1 Glucose-induced insulin release from mouseislets

About 16 years ago both Lenzen

(77)

and Berglund


199

(78)

reported that the insulin secretory responses of theperfused mouse pancreas preparation to glucosestimulation differed significantly from those seenwith the perfused rat pancreas preparation studiedunder comparable conditions. While acute first phaserelease rates were similar, the mouse β-cell responseto glucose stimulation was notable for the absence ofa rising second phase secretory response. Forexample, when rat β-cells were stimulated with highglucose, 30-40 fold increments in insulin release rateswere observed with the perfused pancreas preparation

(7, 8,79)

or from freshly-studied perifused islets


200

(58,65)

(See Figure 1). Second phase responses from mouseislets are flat and from a quantitative perspectivemuch smaller (2-4 fold above prestimulatory rates)than those observed from rat islets

(52)

. This deviation in second phase release from mouseβ-cells has recently been confirmed by Grodsky andcoworkers


201

(80)

, by our group using perifused mouse islets

(52)

and by Sharp and coworkers

(81)


202

studying release from βHC-9 mouse tumoral cells .This difference in glucose sensitivity is unrelated toislet insulin content

(80)

. Of particular importance, the deviation in mouseislet responsiveness to glucose also stands in contrastto the large and rising second phase responses fromhuman islets studied in vivo with the hyperglycemicclamp technique

(10, 12,82)


203

. Thus, while rat and human islets respond tosustained glucose stimulation with a large and risingsecond phase insulin secretory response, mouse β-cells fail to demonstrate this response to glucose.

6.2 PLC activation in mouse isletsIn an attempt to explain the dichotomy in

the insulin secretory responses to glucose stimulationwhich exist between rat and human islets on the onehand and mouse islets on the other, we first focusedour attention on information flow in the PLC/PKCpathway. Detailed glucose dose-response curveslooking at the accumulation of inositol phosphates, asurrogate and highly sensitive marker for PLCactivation, revealed a possible biochemicalexplanation for the anomalous behavior of mouseislets when stimulated by glucose. In response to thehexose, minimal IP accumulation was observed(Figure 2). This suggested to us that at least part ofthe explanation for the smaller secretory effect ofglucose was attributable to the minimal generation ofPI-derived signaling molecules. Since we knew fromour rat islet studies that carbachol activated anisozyme of PLC different from the one activated byglucose, we next measured the IP responses to thisagonist. Comparable stimulatory effects were noted

(54)

and, most importantly, the addition of carbachol orthe protein kinase activator TPA together with 15mMglucose resulted in the emergence of a brisk biphasicinsulin secretory response from mouse islets (Figure4). Before concluding this section it might be positedthat the failure of glucose to stimulate PLC to thesame quantitative extent in mouse islets as in rat, andpresumably human islets, would leave the animal atrisk for hyperglycemia. It must be rememberedhowever that food consumption in these animals isaccompanied by an increase in vagal tone and CCKsecretion. These factors provide the necessary PLC-generated signals to support insulin secretion. Whatmakes human and rat islets so different and unique isthat they have a backup or supplemental system inthat, in addition to neural or incretin factors, fuel ornutrient molecules like glucose are also capable ofproviding significant PLC-generated secondmessengers to reinforce the secretory response.

6.3 Time-dependent potentiation in mouse islets


204

The failure of glucose to evoke a risingsecond phase insulin secretory response from mouseislets when compared to the responses of human orrat islets studied under comparable stimulatoryconditions is not the only anomaly reported to existbetween these species. In follow-up studies Berglund

(83)

reported that prior high glucose stimulation of mouseβ-cells failed to prime or sensitize them torestimulation. This phenomenon, also referred to astime-dependent potentiation (TDP), can be induced inboth rat

(7,16)

and human islets


205

(84,85)

by prior short term glucose exposure. In what mayturn out to be a particularly prescient remark,Berglund

(83)

concluded that the failure of glucose to induce TDPin mouse β-cells may be related to the failure ofglucose to induce a rising second phase secretoryresponse in this species. The fact that the magnitudeof the second phase response may actually regulatethe priming effect of glucose was initially suggestedby Grodsky


206

(4)

25 years ago. The further characterization of thefactor(s) which limit glucose-induced insulinsecretion from mouse β-cells and accounts for theanomalous behavior of mouse β-cells to glucosestimulation when compared to human or rat β-cellsmay offer a most unique opportunity to elucidate thefactors which regulate glucose-induced insulinsecretion. Because of our interest in this process andbecause of our earlier suggestion that both the risingsecond phase response and the induction of TDP seenwhen rat islets are stimulated with glucose may bedependent on events associated with PLC/PKCactivation

(29, 70, 86,87)

, we explored these differences in more detail andattempted to explain these species-dependentresponse patterns to glucose stimulation.

Confirming the findings made with the perfusedpancreas preparation, mouse islets are immune to thesensitizing effect of prior short term glucoseexposure. When briefly exposed to glucose, rat islets


207

Figure 4. Stimulation of Rising Second Phase InsulinSecretory Responses from Mouse Islets. Groups ofislets were isolated from CD-1 mice and perifusedwith 3mM glucose (G3) for 30 min. At this time onegroup was stimulated with 15mM glucose (G15)alone (closed circles), a second group with 15mMglucose plus 500nM of the phorbol estertetradecanoyl phorbol acetate (TPA, open circles),and the third group was stimulated with 15mMglucose plus 100 µM carbachol (closed triangles).Note that while glucose alone is without any effect instimulating a rising second phase, the addition ofcarbachol or TPA together with 15mM glucoseevokes a large, rising second phase response.

respond to subsequent restimulation with adramatically enhanced first phase insulin secretoryresponse

(88)

(See Figure 5) Mouse islets could not be sensitizedunder conditions where rat islets so readily exhibitthis response pattern. If, as we have suggested, thefailure of glucose to activate PLC in mouse islets isthe immediate cause of the failure of the hexose toinduce priming, then it might be suggested thatcarbachol or TPA should be able to induce TDP. Thiswas verified is subsequent studies

(88)


208

(See Figure 6) and further supported the concept thatthe species differences in the expression andactivation of PLC played a major and determiningrole in the species-dependent response patterns toglucose stimulation.

6.4 Glucose fails to induce time-dependentsuppression (TDS) in mouse islets

While the positive stimulatory actions ofglucose on the β-cell have been the primary foci upuntil now, it is also clear that sustained exposure tothe hexose impairs insulin secretion. Termed TDS, itcan also be induced by other agonists including butnot confined to those compounds which act initially toincrease information flow in the PLC/PKC signalingsystem. Thus, TDS can be induced by long term (2-3hour) exposure to cholecystokinin (CCK) or tocarbachol as well as by sustained exposure toforskolin, which elevates cAMP to supraphysiologicallevels in islets

(89)

. In islets suppressed by any one of these compounds,the common biochemical lesion and the one whichappears to account for reduced insulin secretion is theimpaired capacity of subsequent restimulation withglucose to activate PLC. Our working hypothesis toaccount for the induction of TDS is that the sustainedactivation of PKC or PKA results in thephosphorylation of PLC, a type of negative feedbackand an event which reduces PLC's ability tosubsequently respond to stimulation. This proposedmodel of inhibition is consistent with what has beenobserved in other systems as well: PKC activationinhibits PLC activation

(53, 90,91)


209

. Moreover, PLC activation is also inhibitable bycAMP as well, providing an explanation for theinhibitory action of forskolin on β-cell PLC and itscapacity to induce TDS of insulin release. If sustainedPLC/PKC activation by high glucose is responsiblefor the induction of TDS of secretion in rat islets,then mouse islets which respond poorly in terms ofPLC-mediated PI hydrolysis should be relativelyimmune to the desensitizing effect of the hexose. Thishas been confirmed using perifused mouse isletspreviously exposed to 20mM glucose for 3 hr

(55)

(See Figure 7). For example, 3 hour exposure of ratislets to 20mM glucose impaired the capacity of theseislets to respond subsequently to stimulation with20mM glucose plus 100µM carbachol; 20mMglucose induced TDS of release. Mouse isletssecretory responses studied after a 3 hour incubationperiod with 20mM glucose were not suppressed whenstimulated with 20mM glucose plus 100µMcarbachol; 20mM glucose failed to induce TDS ofrelease in mouse islets. Studies with mouse isletshave afforded us a most unique opportunity to dissectout the contribution of glucose signaling via thePLC/PKC cascade to the regulation of insulinsecretion.

7. THE BETA CELL, OBESITY AND NIDDM

Before summation it is perhaps notunreasonable to place the basic science studiesdiscussed above into some type of clinicalperspective. The question might be posed as to howsignaling in the PLC/PKC system may be involved inaltered patterns of insulin secretion established tooccur in both obesity and NIDDM. In obesity, β-cellsecretion is dramatically amplified. Thus, in bothhuman obesity


210

(92,93)

and in several well characterized animal models ofobesity

(94,95)

, early and perhaps even primary changes in β-cellsensitivity to stimulation have been observed. In fact,even in preobese mice, the sensitivity of their islets toagonists which increase information flow in thePLC/PKC signaling system is enhanced andcontributes to the early and perhaps primaryhyperinsulinemia in these animals


211

(96)

. Since insulin resistance can be acquired as aconsequence ofhyperinsulinemia

(97-99)

, changes at the level of β-cell signal transductionsystems which amplify their stimulatory effect oninsulin release, may be primary etiologic componentsin the odyssey toobesity


212

(100)

. Finally, while there is little doubt that β-cell failure

Figure 5. Glucose Stimulation Induces Time-Dependent Potentiation in Rat but not Mouse Islets. Groups of rat (left, open circles) ormouse (right, open circles) islets were perifused for 30 min with 3mM glucose (G3) followed by 15 min with 15mM glucose (G15). Aftera 15 min washout with 3mM glucose they were stimulated with 15mM glucose for 30 min and this is the period shown. The responses ofcontrol islets (closed circles) perifused for 60 min with 3mM glucose prior to 15mM glucose stimulation are also shown. Note that priorglucose sensitized rat but not mouse islets. Note also the change in scale between panels.


213

Figure 6. Prior Exposure to the Phorbol Ester TPA Induces Time-Dependent Potentiation in Mouse Islets. Two groups of mouse isletswere studied. One group (open circles) was perifused for 60 min with 3mM glucose (G3) prior to stimulation with 15mM glucose (G15).The second group (closed circles) was perifused for 30 min with 3mM glucose, 15 min with 3mM glucose plus 500nM TPA and for anadditional 15 min with 3mM glucose alone. They were then stimulated with 15mM glucose and this is the period shown here.

Figure 7. Effects of Prior 3hr. Exposure to 20mM Glucose on Insulin Release Rates from Rat or Mouse Islets: High Glucose Fails toInduce Suppression in Mouse Islets. Groups of islets were isolated from Sprague-Dawley (SD) rats (left) or CD-1 mice (right) andincubated for 3 hr in 5mM glucose alone (closed circles, solid line) or 20mM glucose alone (closed triangles, dashed line). All groups ofislets were then perifused for 30 min with 5mM glucose (G5) and for an additional 30 min with the combination of 20mM glucose (G20)plus 100µM carbachol (Carb). This combination of agonists was used because glucose alone is such a poor stimulant for secretion frommouse islets. Note the reduction in release from rat islets due to prior 20mM glucose exposure but the slight increase from mouse islets.

or decompensation is essential in the development offrank NIDDM, the nature of the biochemical lesionresponsible for the reduction in insulin secretion isnot known. However, since β-cell failure to secreteenough insulin to maintain glucose homeostasis is aconsequence ofhyperglycemia

(32)


214

and since high glucose desensitizes the β-cell byinhibiting PLC activation

(33,101)

, a reasonable suggestion is that the impairedactivation of β-cell PLC may precipitate NIDDM insome individuals.

9. SUMMARY AND PERSPECTIVE

Because of the essential role of insulin inthe maintenance of fuel homeostasis, the role of thesignal transduction systems which regulate β-cellsecretion of the hormone assume physiologic andclinical significance. In the present review, we haveattempted to draw the readers attention to the key andessential role of information flow in the PLC/PKCsignal transduction pathway in determining how theβ-cell works and why it may fail or decompensate.Results from studies support the concept that thissystem is involved in glucose-induced biphasicinsulin secretion, in particular the rising second phaseresponse, the induction of TDP, characterized by amarkedly enhanced first phase response, and theinduction of TDS where insulin secretory failureoccurs. Studies from several labs have demonstratedmarked species differences between rat and mouse β-cell sensitivity to glucose stimulation, differences wehave attributed to a dichotomy in the informationflow in the PLC/PKC signaling system. Finally, thepotential role of this transduction system in theetiology of hyperinsulinemia, insulin resistance,obesity and, all too often, in NIDDM has beensuggested. Along these same lines, it would be ofparticular interest to ascertain how overexpression ofPKC influences glucose homeostasis and whethertreating mouse islets with viral vectors containing thecDNAs for the various isozymes of PLC, as has beendone with otherenzymes


215

(102)

, converts their response patterns to those reminiscentof rat islets. Finally, in attempts to geneticallyengineer pancreatic β-cells with an eye toward theirtransplantation, consideration will have to be given tothe content and expression of the PLC isozymes andPKC in these cells if the physiologic regulation ofinsulin secretion is to be duplicated.

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